Stimulation Of Bradykinin B2 Receptor Research Articles

Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene

Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3–protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and β-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.

Phosphorylation of epidermal growth factor receptor at serine 1047 in cultured lung alveolar epithelial cells by bradykinin B2 receptor stimulation

Accumulating evidence indicates that epidermal growth factor receptor (EGFR) is desensitized by phosphorylation of serine 1047 (Ser1047). We and other groups have reported that stimulation of a receptor of tumor-necrosis factor α (TNFα) and Toll-like receptor 5 (TLR5) induced the phosphorylation of Ser1047 through activation of p38 mitogen-activated protein kinase (p38 MAPK) in cultured lung alveolar epithelial A549 cells. However, phosphorylation of EGFR at Ser1047 by stimulation of any G-protein coupled receptors (GPCRs) has not been reported in any cultured cells. In the present study, we first confirmed that A549 cells expressed bradykinin (BK) B2 receptor, and then, we examined whether BK treatment of A549 cells activated MAPKs and induced the phosphorylation of EGFR at Ser1047. Immunoblotting analysis and reporter gene assays indicated that BK activated the pathways of extracellular signal-regulated kinase (ERK) and p38 MAPK. Inhibitor studies suggested that Gq/11 was mainly involved in the activation of ERK and p38 MAPK. We found that stimulation of the BK B2 receptor, but not the BK B1 receptor, induced phosphorylation of EGFR at Ser1047. Pharmacological experiments indicated that both ERK and p38 MAPK were involved in the phosphorylation of EGFR. These results strongly suggested that BK regulates EGFR functions in lung alveolar epithelial cells. In addition, we found that BK treatment increased the mRNA level of dual specificity MAPK phosphatase 5 (DUSP5) in an ERK-dependent manner, which suggested that a negative feedback mechanism of ERK existed in the cells.

Intratesticular Bradykinin Involvement in Rat Testicular Pain Models.

To clarify the role of bradykinin in urogenital pain, we investigated bradykinin involvement in rat models of testicular pain. Bradykinin (0.1, 0.3, 1, 3 and 10 mmol/L) or distilled water was injected into the testes of male Wistar rats, and induced pain behaviors in conscious rats were evaluated. The effect of pretreatment with bradykinin B2 receptor antagonist FK3657 on bradykinin-induced pain behavior was then examined. We also evaluated the analgesic effect of FK3657 in a rat acetic acid-induced testicular pain as well as changes in the intratesticular bradykinin concentration after testicular injection of acetic acid. An injection of bradykinin into the testes of conscious rats induced pain behaviors that were dose-proportionally reduced by prior administration of FK3657. In addition, FK3657 dose-dependently inhibited the pain responses induced by testicular injection of 1% acetic acid. An increase in intratesticular bradykinin concentration was detected after the testicular injection of 1% acetic acid. Here, we found that intratesticular bradykinin evokes pain behavior via stimulation of bradykinin B2 receptors and that intratesticular acetic acid injection induces intratesticular bradykinin synthesis, consequently leading to pain behavior. These findings suggest that the potential utility of bradykinin B2 receptor antagonists as a novel target for treating urogenital pain.

Novel Insights Into the Critical Role of Bradykinin and the Kinin B2 Receptor for Vascular Recruitment of Circulating Endothelial Repair–Promoting Mononuclear Cell Subsets

Background— Endothelial injury is considered critical for progression of atherosclerosis and its complications in coronary artery disease (CAD). The endothelial-supportive effects of bradykinin have mainly been attributed to activation of the resident endothelium. Here we newly investigate the role of bradykinin and its B2 receptor for the recruitment and functional activation of circulating mononuclear cell subsets with endothelial-repair promoting capacity, such as CD34 + CXCR4 + cells, at sites of arterial injury. Methods and Results— Bradykinin-B2-receptor (B2R) blockade by icatibant substantially impaired recruitment of circulating CD34 + CXCR4 + mononuclear cells (expressing high levels of B2R) to endothelial cells in vitro and to injured arterial wall in vivo, whereas recruitment of CD14 hi monocytes (expressing low levels of B2R) was unchanged. Moreover, the capacity of genetically B2R -deficient bone marrow cells to promote endothelial repair in vivo was markedly impaired as compared with wild-type bone marrow cells. B2R expression was reduced on CD34 + CXCR4 + mononuclear cells and endothelial repair–promoting early outgrowth cells, but not on CD14 hi monocytes, from CAD patients as compared with healthy subjects. B2R stimulation induced CD18 activation in early outgrowth cells of healthy subjects, but not in early outgrowth cells of CAD patients. Adenoviral B2R overexpression enhanced in vivo vascular recruitment and rescued impaired endothelial repair capacity of early outgrowth cells from CAD patients. Conclusions— We newly report that bradykinin/B2R signaling may promote endothelial repair after arterial injury by selective recruitment and functional activation of B2R-expressing circulating mononuclear cell subsets. In CAD patients, B2R downregulation on endothelial repair–promoting circulating mononuclear cells substantially impairs the bradykinin-dependent endothelial repair, representing a novel mechanism promoting endothelial injury in CAD patients.

Involvement of Bradykinin in Trypsin-Induced Urinary Bladder Contraction in Cyclophosphamide-Treated Rats

Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Previous studies have demonstrated that both trypsin and PAR-2 activating peptide contract isolated rat urinary bladder preparations, however, the mechanisms are not fully understood. In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH(2) in urinary bladders isolated from control or cyclophosphamide (CYP)-induced cystitis rats. The contractile effects of trypsin were significantly greater in the preparations obtained from CYP-treated rats than in those from controls. The bradykinin B2 receptor antagonist Hoe 140 did not affect trypsin-induced contractions in control rat bladders, whereas it significantly reduced the contractile effects of trypsin on bladders from CYP-treated rats. On the other hand, Hoe 140 failed to affect contractions induced by the PAR-2 agonist 2-furoyl-LIGRL-NH(2). These results suggest that the actions of trypsin on urinary bladders in cystitis rats are partly exerted through stimulation of bradykinin B2 receptor in a PAR-2-independent manner. This mechanism seems to be involved in the enhancement of trypsin-induced bladder contractions observed after induction of cystitis with CYP in rats.

Intracellular Ca2+ Increase in Neuro-2A Cells and Rat Astrocytes Following Stimulation of Bradykinin B2 Receptor

Murine neuroblastoma cell line Neuro-2A cells and rat brain astrocytes showed a dose-dependent increase in intracellular Ca2+ in response to bradykinin, when assessed by a single cell image analyzing system. The Ca2+ increase in Neuro-2A cells by bradykinin was also examined by a suspension fluorescent assay using fura-2 loading. The Ca2+ increase in both cases was suppressed by a bradykinin B2 receptor antagonist, Hoe 140, but not by a B1 receptor antagonist, des-Arg-Hoe 140, suggesting that the effect occurred via specific B2 receptor activation. RT-PCR for bradykinin B2 receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B2 receptor mRNA. Binding of [3H]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75 nM was determined. Furthermore, the increase in [Ca2+]i by bradykinin could be caused by a release of Ca2+ from storage sites in the endoplasmic reticulum, since thapsigargin and U-73122 attenuated the effect of bradykinin in Neuro-2A as well as in astrocytes. These results indicate that both astrocytes and neuroblastoma Neuro-2A cells stimulated by bradykinin could express a bradykinin B2 receptor-mediated intracellular Ca2+ increase leading to signal transduction.

Bradykinin Regulates Captopril-induced Upregulation of β-Adrenergic Receptor in Cultured Neonatal Rat Cardiomyocytes

S. Yasunaga, H. Yonemochi, T. Saikawa and T. Sakata. Bradykinin Regulates Captopril-induced Upregulation of β-Adrenergic Receptor in Cultured Neonatal Rat Cardiomyocytes, Journal of Molecular and Cellular Cardiology (2000)32 , 153–159. The aim of this study was to elucidate the role of bradykinin in mediating captopril-induced upregulation of β -adrenergic receptor (β -AR). The density of β -AR on the surface of cardiac myocytes was measured by binding assay using [3H]CGP-12177. Treatment of cultured neonatal rat cardiomyocytes with captopril resulted in a time-dependent elevation of bradykinin concentration in the culture medium. The increased bradykinin concentration was significant at 2, 3 and 6 h, but not at 12 h after exposure to captopril. This time-dependent effect of captopril on enhancement of bradykinin levels paralleled that of β -AR upregulation. Exogenously applied bradykinin increased β -AR density by 22, 30 and 35% at 0.01, 0.1 and 1 μ mconcentrations, respectively. Myocytes treated with 1 μ m bradykinin responded to isoproterenol (ISP) in a dose-dependent manner, as demonstrated by acceleration of spontaneous beating frequency. These beating acceleration effects of bradykinin were abolished by Hoe 140. Stimulation of bradykinin B2 receptor by exogenously added bradykinin for 6 h was sufficient to produce β -AR up-regulation to a level similar to that seen after 24 h. Our results indicate that bradykinin potentiation by ACE inhibitors regulates, at least in part, captopril-induced β -AR up-regulation.

Endogenous B1 receptor mediated hypotension produced by contact system activation in the presence of endotoxemia

Previous studies have shown that an intravenous infusion of dextran sulfate (DXS) causes arterial hypotension via release of bradykinin (BK) and stimulation of bradykinin B2 receptors in pigs. The bradykinin B1 receptor is not physiologically present but its expression can be induced by bacterial lipopolysaccharide (LPS). This study was designed to assess the relative roles of bradykinin B2 and B1 receptors in the hypotensive response produced by DXS in LPS-treated pigs. In LPS-treated pigs a continuous infusion of DXS produced a progressive drop in blood pressure that peaked at approximately 30 min after onset of the infusion and returned to baseline after another 30 min. In animals receiving the selective B2 receptor antagonist Hoe-140 a significant attenuation of the peak fall in blood pressure to DXS was observed. In pigs treated with Hoe-140 and the selective B1 receptor antagonist CP-0298 (Lys0-Leu8-des-Arg9-bradykinin) DXS infusion had no effect on blood pressure. This is the first demonstration in vivo that following activation of the contact system both B2 and B1 receptors are involved in the resulting hypotensive response. This would be consistent with the production of BK (which stimulates B2 receptors) that is subsequently converted to the biologically active metabolite des-Arg9-BK in sufficient concentrations to activate B1 receptors. The significance of these observations to pathophysiology remains to be determined.

Pharmacology and mode of action of bradykinin on mouse-isolated trachea.

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.

Ca2+‐dependent and ‐independent mechanism of cyclic‐AMP reduction: mediation by bradykinin B2 receptors

1. Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2. The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3. The Ca(2+)-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4. The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5. Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca(2+)-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6. The bradykinin B1 receptor agonist, desArg9-bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7. Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK. The bradykinin B2 receptor agonist,desArg9-bradykinin, did not evoke a rise in cytoplasmic Ca2 .8. It is concluded, that stimulation of bradykinin B2 receptors causes a reduction in cellular cyclic AMP in DDT1, MF-2 cells. This decline in cyclic AMP is partly mediated by a Ca2+/calmodulin independent activation of phosphodiesterase activity. The increase in [Ca2+], mediated by bradykinin B2 receptors inhibited forskolin- and isoprenaline-activated adenylyl cyclase differently, most likely by interfering with different components of the adenylyl cyclase signalling pathway.

Role played by NK2 receptor and cyclooxygenase activation in bradykinin B2 receptor mediated-airway effects in guinea pigs.

We have investigated the effects of SR-48968, an NK2 receptor antagonist, and indomethacin, a cyclooxygenase inhibitor, against bronchoconstriction and airway microvascular leakage induced by bradykinin (BK) in anesthetized guinea pigs. In addition, we have determined whether these effects were mediated via bradykinin B2 receptor activation, using a B2 receptor antagonist HOE 140. Lung resistance (RL) and extravasation of Evans blue dye into airway tissues were used as indexes of airway caliber and microvascular leakage, respectively. BK (15 nmol/kg i.v.) induced a significant increase in RL and leakage of dye at all airway levels, responses which were completely abolished by HOE 140 (0.13 mg/kg i.v.). SR-48968 (1.5 mg/kg i.v.) had no effect against BK-induced airway effects. Indomethacin (5 mg/kg i.v.) completely blocked the increase in RL and significantly inhibited the leakage of dye in peripheral intrapulmonary airway. In conclusion, bronchoconstriction induced by i.v. BK is mediated by release of cyclooxygenase products but not by stimulation of NK2 receptors, while the airway microvascular leakage only partly involves cyclooxygenase activation. Cyclooxygenase activation may occur following bradykinin B2 receptor stimulation.